The retinoblastoma tumor suppressor (pRB) and two related proteins (P107 and P130; collectively referred to as pocket proteins) playa fundamental role in regulating proliferation, cell cycle progression, and cell cycle exit. pRB has also been shown to play an essential role in differentiation of several tissues, including muscle. Nevertheless, our understanding of the regulatory controls involving the transition from a proliferative to a quiescent and, finally, terminally differentiated state is incomplete. Our work during the previous funding period has focused on the unique roles for each of these pocket proteins in responding to growth arrest cues and in both promoting and maintaining differentiation of muscle. In addition, we have identified transcriptional and epigenetic regulatory mechanisms associated with cell cycle exit and terminal differentiation. In particular, we have begun a systematic investigation of all chromatin modifications associated with myogenic differentiation using a combination of ChIP-sequencing (ChIP-seq) and expression profiling. Here, we propose the following aims to further dissect the mechanisms underlying pocket protein involvement in cell cycle exit and differentiation. In our first Aim, we will examine gene regulation during myogenic differentiation by specifically focusing on the role of histone H2B ubiquitylation (H2BUb) during differentiation. We will examine the extent of histone cross-talk in myotubes and investigate the impact of ablating RNF20, the enzyme responsible for H2B ubiquitylation, on differentiation. Remarkably, we have found that H2BUb essentially disappears during differentiation, and therefore we will examine the underlying mechanistic basis for myotube-specific loss of H2Bub. In our second Aim, we will examine the genome-wide role of pRB and co-repressors in directing chromatin modifications. We will first perform ChIP-seq on pRB and P130 in differentiated myotubes, enabling us to identify regions bound by these factors. We will also perform expression profiling after removal of pRB or P130 from myotubes. By merging these data, we will determine how chromatin modifications and gene expression are affected by loss of pocket proteins. Using our extensive ChIPseq data as a guide, we will test the hypothesis that pRB directs tri-methylation of H3K27 (H3K27IDe3) and ask whether Polycomb repressive complex (PRC2) is involved and to what extent. We will examine whether Ezh2 or an alternative histone methyltransferase (HMT), such as Ezhl, may be involved in H3K27me3 deposition. We will determine whether pRB influences the recruitment of either or both HMTs. The acquisition of our extensive ChIP-seq dataset allows us an unprecedented ability to examine on a genome-wide scale the relationship between pRB binding, chromatin modifications, and gene expression in a developmentally relevant setting, thereby enhancing our understanding of regulatory controls that are essential for both reversible and permanent gene silencing, withdrawal from the cell cycle, and muscle differentiation.